Activin antagonists as novel contraceptives

ABSTRACT

A method for contraception which features administering to a patient a contraceptive agent having activin-antagonist activity. The contraceptive agent results in inhibition of the activin-stimulated release of FSH, thereby resulting in a decrease in fertility and facilitating contraception in the patient.

The invention described in this application was supported by theNational Center for Infertility, research grants HD29164 and HD28138.The U.S. government has certain rights in this invention.

This invention relates to contraception.

This is a continuation of application Ser. No. 08/234,261, filed Apr.28, 1994 now U.S. Pat. No. 5,658,876.

BACKGROUND OF THE INVENTION

Release of FSH from the anterior pituitary is essential forgametogenesis, as FSH results in stimulation of spermatogenesis in thetestis, as well as maturation of the oocyte and secretion of estradiolby the ovarian follicles. The biosynthesis and secretion of FSH withinthe anterior pituitary is controlled through the complex interaction ofseveral hormones. These include hypothalamic gonadotropin-releasinghormone (GnRH), gonadal steroids, and the recently-identified gonadalpeptides, inhibin, activin, and follistatin.

Inhibin and follistatin have the ability to inhibit the synthesis andsecretion of FSH by the pituitary (De Jong et al., Nature 263: 71-72(1976)), while activin has been shown to be capable of stimulating FSHsynthesis and release (Vale et al., Nature 321: 776-779 (1986); Ling etal., Nature 321: 779-782 (1986)). Inhibin is a heterodimericglycoprotein composed of an α subunit linked to one of two β subunits(β_(A) or β_(B)), whereas activin consists of heterodimers or homodimersof inhibin β subunits. Although originally isolated from the gonads,both inhibin and activin have been detected in the pituitary where theyplay an autocrine/paracrine role in the control of FSH gene expression(Meunier et al., Proceedings of the National Academy of Sciences85:247-251, (1988)). In addition, recent immunohistochemical studieshave determined that inhibin/activin subunit proteins localized in thepituitary gonadotropes are responsible for FSH and LH synthesis (Robertset al., 1989, Endocrinology 124:552-554).

Both activin and inhibin subunits have been isolated from naturalsources. In Mason et al., U.S. Pat. No. 4,798,885, the amino acidsequences of the α, β_(A) and β_(B) subunits of human and porcineinhibin are described and are used to identify the amino acid sequencesof activin. Mason et al. discuss the use of activin as afertility-inducing therapeutic capable of stimulating FSH release.

The use of activin to increase fertility in a male mammal is describedin Attie et al., PCT No. 91/10444. Following treatment of rat testiculargerm cells with activin, these cells were observed to proliferate2.0-2.5 times faster than control cells. Additional studies haveconfirmed the initial description of activin's ability to stimulate FSHsecretion from cultured rat pituitary cells by about 2-3 fold (Vale etal., Nature 321: 776-779 (1986); Ling et al., Nature 321: 779-782(1986); Carroll et al., Molecular Endocrinology, 3:1969-1976 (1989);Kowaga et al., Endocrinology 128, 1682-1684 (1991); Attardi et al., Mol.Endo. 4: 721 (1990)). Although these results raise the possibility thatactivin may increase fertility in mammalian subjects, the measuredincrease in FSH secretion is typically small.

Recent results suggest that the importance of activin to maintenance ofFSH biosynthesis is much greater than has previously been recognized.The mRNA for the β subunits of FSH become rapidly undetectable inpituitary cells cultured in a way to remove endogenous-secreted activin(Weiss et al., Endocrinology 131: 1403-1408 (1992)). When activin isthen added back to the medium, these low levels of FSH mRNA increase30-70 fold, a substantially greater increase than the 2-3 foldstimulation that is observed when cells are cultured in the presence ofendogenous activin.

The biological activity of FSH can be influenced by the presence of abinding protein, such as follistatin (FS) (Ling et al., Nature 321:779-782 (1986)), which may form complexes with activin (Krummin et al.,Endocrinology 132: 431 (1993)). Recent studies indicate that incubationof rat pituitary cultures with an immunoneutralizing antibody raisedagainst activin results in a decline in FSH secretion (Corrigan et al.Endocrinology, 128:1682-1684 (1991)), and that FS acts similarly bybinding to activin, thereby neutralizing its FSH-releasing activity(Kowaga et al., Endocrinology 128, 1682-1684 (1991)).

SUMMARY OF THE INVENTION

In general, the invention features a method for contraception byinhibiting the synthesis and/or release of FSH in a patient, the methodfeaturing the step of administering to the patient a contraceptive agenthaving activin-antagonist activity such that the contraceptive agentresults in inhibition of the activin-stimulated synthesis and/or releaseof FSH, resulting in a decrease in fertility and facilitatingcontraception in the patient.

In preferred embodiments of the invention, a peptide or non-peptidemimetic having activin-antagonist activity is used as the contraceptiveagent. The amino acid sequence of the peptide or chemical structure ofthe non-peptide mimetic is sufficiently homologous with the receptorbinding region of the human/activin β subunit such that it caneffectively bind to the activin receptor sites and inhibitactivin-stimulated release of FSH.

In a particular embodiment, the peptide contains between 8 and 70 aminoacids, preferably a region at least 8 amino acids in length, and morepreferably 15 amino acids in length, which is at least 80% homologouswith a portion of the binding region of the activin β subunit;preferably, the portion of the binding region includes the N-terminal orC-terminal 8 amino acids of the binding region.

In particular embodiments of the invention, the peptide contains anamino acid sequence selected from the group consisting of:

Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp Gly Gln (SEQID NO:1) (amino acids 82-98 of the activin β_(A) subunit)

and

Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val Glu Glu Cys Gly (SEQ IDNO:2) (amino acids 99-114 of the activin β_(A) subunit), or asubstantially homologous variant thereof. In a preferred embodiment, thepeptide includes an amino acid sequence substantially homologous to thesequence:

Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp Gly Gln AsnIle Ile Lys Lys Asp Ile Gln Asn Met Ile Val Glu Glu Cys Gly (SEQ IDNO:3) (amino acids 82-114 of the activin β_(A) subunit).

In another embodiment of the invention, the contraceptive agent includesa protein having activin-antagonist activity which is capable of bindingto activin to inhibit activin-stimulated release of FSH. In a particularembodiment, the protein is FS or a protein substantially homologous toFS.

By "substantially homologous" is meant an amino acid sequence whichdiffers from a region of the activin β subunit only by conservativeamino acid substitutions, for example, substitution of one amino acidfor another of the same class (e.g., valine for glycine, arginine forlysine, etc.) or by one or more non-conservative substitutions,deletions, or insertions located at positions which do not destroy thebinding function of the peptide, but confer greater stability andantagonist activity. Preferably, a peptide is greater than 80%homologous (i.e., greater than 80% identical) to a region of the activinβ subunit.

In yet another preferred embodiment of the invention, the contraceptiveagent features a modified activin receptor having activin-antagonistactivity which is capable of both binding activin and inhibitingactivin-induced signaling once activin is bound, resulting in inhibitionof activin-stimulated release of FSH. Alternatively, the modifiedactivin receptor can attach to an activin receptor to form a complex.The complex is capable of binding activin, and inhibits activin-inducedsignaling once activin is bound, resulting in inhibition ofactivin-stimulated release of FSH. In preferred embodiments, the complexis a dimer.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a contrast image showing the stimulation of FSH mRNA levels byactivin or pulsatile GnRH in perifused pituitary cells.

FIG. 2 is a plot showing gonadatropin subunit mRNA levels as a functionof activin concentration in perifusion experiments.

FIG. 3 is a plot showing the molecular weight shift in activin-FScompounds compared to unbound activin in buffer.

FIG. 4 is a plot showing the specific binding of activin and inhibin asa function of FS concentration.

FIG. 5 is a plot showing the percentage of activin stimulated as afunction of FS concentration.

FIG. 6 is a plot showing the inhibition of activin's interaction with FSby several synthetic peptides due to overlap with FS-activin bindingregion.

FIG. 7 is a plot showing the dependence of FSH release on activin, FS,and activin-FS complexes.

FIG. 8 is a plot showing the dependence of FSH release on the syntheticpeptide BIN-3 (SEQ ID NO:3).

FIG. 9 is a plot showing the dependence of FSH release on BIN-2 (SEQ IDNO:2) and BIN-3 (SEQ ID NO:3) in the presence of activin.

DETAILED DESCRIPTION

The method of providing contraception by antagonizing the effects ofactivin was in part motivated by Applicants' discovery that activin is amore potent activator of FSH β subunit mRNA production than previouslydescribed. Using an assay involving the continuous infusion of activinin cultured pituitary cells, it was determined that activin increasedlevels of FSH mRNA production by 30-70 fold, a level more than an orderof magnitude greater than that previously reported. In animals, FSHsecretion results in stimulation of spermatogenesis in the testis andsecretion of estradiol by the ovarian follicles, thus the inhibition ofactivin-stimulated FSH release is an effective method for contraception.

Activin-stimulated release of FSH can be inhibited by a number ofdifferent mechanisms. Hormone binding proteins which compete for bindingto the activin receptor are shown to regulate the bioactivity ofactivin. Binding proteins which form complexes with activin may alsoeffect the release of FSH. One example of such a protein is FS, whichcan bind directly to activin, thus reducing its bioactivity. The actionof activin can also be regulated by modifying the receptor involved withbinding. Receptors can be constructed so that the binding andmembrane-spanning domains are the same as those of the normal activinreceptor, but the intracellular sequences required for signalling areabsent. In this case, the binding of activin to the receptor isuninhibited, but the activin-stimulated signalling of the receptor iseffectively blocked.

There now follows a description of the effect of activin antagonists onthe secretion of FSH, and the methods used to make such antagonists.

Regulation of FSH mRNA Levels by Activin in Perifusion Columns

The experiments described below used to investigate the regulation andsecretion of FSH subunit mRNA levels by activin and GnRH are describedin Weiss et al., Endocrinology 131: 1403-1408 (1992).

Pituitary cells were cultured using a perifusion column which allowedcontinuous infusion of activin and removal of endogenously secretedactivin from the culture, resulting in activin-enhanced stimulation ofFSH mRNA levels in perifused pituitary cells which is an order ofmagnitude greater than the response seen previously in static cellcultures (Kowaga et al., Endocrinology 128, 1682-1684 (1991); Attardi etal., Mol. Endo. 4: 721 (1990)). The effects of pituitary-derived factorsare minimized by continuous perifusion, making it possible to assess thesensitivity of unstimulated cells to exogenously-administered activin.For example, the response of cells in static culture to activin may bepreempted by the stimulatory effects of endogenously produced pituitaryfactors. These factors would be rapidly removed from perifusion columnsbut appear to accumulate in culture dishes and mask the effects ofexogenous stimulation.

For the experiments described hereafter, cells were perifused withactivin, cultured, and analyzed as described previously (Weiss et al.,Endocrinology 131: 1403-1408 (1992); Weiss, et al., Endocrinology127:2364-2371 (1990)).

Gonadotropin Subunit mRNA Levels After Activin Treatment

In an initial experiment, the response of FSH mRNA to recombinantactivin was compared with the response to GnRH, a known hypophysiotropicstimulator of FSH mRNA levels (Gharib, et al., Endocrine Reviews,11:177-199 (1990)). The comparison was made between pituitary cellsprocessed in a perifusion column, allowing the dual advantage ofcontinuously removing endogenously produced factors and allowing theadministration of a pulsatile signal, which is critical for GnRHstimulation of FSH expression (Weiss, et al., Molecular Endocrinology,4:557-564 (1990)). Cells were prepared for perifusion as describedpreviously (Weiss, et al, Mol. End. 4:557-564, (1990)). Individualcolumns were perifused for 10 hrs with either perfusion medium(RPMI-1640 containing 2 g/l NaHCO₃, 0.25% BSA (Fraction V; Sigma, St.Louis, Mo.)) alone, hourly pulses of 10 nM GnRH, or a continuousinfusion of a maximal concentration of activin (50 ng/ml). At the end ofthe experiment, total RNA was prepared and quantitated by a northernblot analysis described previously (Weiss, et al, Mol. End. 4:557-564,(1990)).

Referring now to FIG. 1, FSH levels were increased following exposure toGnRH and activin. In the experiment, GnRH was administered as hourly 5min. pulses and activin was administered continuously over 10 hrs.,while the control column received perifusion medium alone. Applicationof pulsatile GnRH elicited a 2.7-fold increase in the level of FSH mRNAcompared to application of the perifusion medium alone. More strikingwas the response of FSH mRNA to activin in perifusion; the observed55-fold increase in FSH mRNA levels was considerably larger than thepreviously reported effects of activin in plated cell cultures.

The disparity in response between perifused cells and cells cultured indishes was examined further using a range of activin concentrations.Referring now to FIG. 2, activin concentrations exceeding 0.03 ng/mlresulted in an increase in FSH levels during perifusion experiments. FSHsubunit mRNA levels were calculated as a ratio to GAPDH, and wereexpressed in terms of fold change relative to columns receivingperifusion medium alone. Molar concentrations of activin were calculatedfrom mass amounts assuming a molecular weight of 28000 g/mol. forrecombinant activin. Results were confirmed using additional experimentsinvolving perifusion medium alone and medium containing concentrationsof activin ranging from 0.01 ng/ml to 10 ng/ml. In all cases, activinwas exceptionally effective and highly specific for stimulation of FSHmRNA levels.

Referring again to FIG. 2, a detectable increase in the level of FSHmRNA was measured using 0.03 ng/ml of activin (1.79-fold, not clear onthis scale), and stimulation exceeded 10-fold at 0.1 ng/ml, whichapproximates the ED₅₀ (0.12 ng/ml) for activin in this system. 25-foldstimulation was achieved at 1 ng/ml activin, with higher concentrationseliciting no additional response in this experiment. In additionalexperiments, elevations of FSH mRNA levels in response to higherconcentrations of activin (3 ng/ml) ranged from 20-fold to greater than75-fold.

For comparison, a concentration of activin (5 ng/ml) that maximized FSHlevels in the perifusion system was tested in plated cell cultures.Cells were cultured for 3 days and then exposed to fresh medium with andwithout activin for 72 hours. In the experiment, mRNA levels werecalculated and expressed as described for FIG. 2. Under theseconditions, activin was considerably less effective in stimulating therelease of FSH, eliciting only a 2.7-fold increase in the mRNA level.

Time-Course of FSH mRNA Expression After Pituitary Cell Dispersion

The continuous flow of fresh medium through perifusion columns removessecreted factors (released by the perifused cells) which normallyaccumulate in static culture dishes. To examine the possibility thatendogenous factors were stimulating pituitary cells in plated cellcultures and thereby preventing the full response to exogenous activin,the dynamic changes in FSH mRNA levels from the time of pituitarydispersion were monitored in culture dishes during a 3-day period. TotalRNA was extracted from equal cell aliquots immediately after dispersionat different times after being placed into culture dishes. Expression ofFSH mRNA fell very rapidly after dispersion, declining to 8% of itsoriginal level at 4 hrs, and remained low (15% of the original level) 8hrs after dispersion. The level of FSH mRNA increased to 68% of theoriginal level 24 hrs after dispersion, and continued to rise, exceedingtwice the immediate post-dispersion level by 3 days.

Interference of FSH Biosynthesis

Release of FSH from the anterior pituitary is essential forgametogenesis, and results in stimulation of spermatogenesis in thetestis and secretion of estradiol by the ovarian follicles. Methods ofantagonizing the activin-stimulated release of FSH in a patient are usedin the present invention as a way of providing contraception.

In one method of the invention, FSH biosynthesis can be blocked usingmolecules which compete for the activin receptor, thus inhibitingactivin-induced release of FSH. Alternatively, compounds which formcomplexes directly with activin, such as FS, can block activin'sbiological activity without actually competing for the activin receptorsite. Another method of the present invention used for inhibitingactivin-induced release of FSH involves using a modified activinreceptor as the activin antagonist. It is possible to construct anactivin receptor which lacks the intracellular sequences required forsignaling, but has binding and membrane spanning domains capable ofbinding activin. In this case, activin binding is not affected, butactivin signalling is inhibited, resulting in a decrease inactivin-induced FSH biosynthesis.

The methods of contraception of the present invention using activinantagonists are carried out as follows.

EXAMPLE 1 FS as an Activin Antagonist

Inhibition of activin's biological activity through binding with FS wasmonitored with the following experiments. Radiolabelled activin wasincubated with serum samples and chromatographed on a gel filtrationcolumn, resulting in two peaks corresponding to FS-activin complexes.Each peak corresponded to a higher apparent molecular weight than thepeaks observed for activin chromatographed in buffer alone. Referringnow to FIG. 3, 100 ng FS bound all of the radiolabelled activin,resulting in a sharp peak eluting with M_(r) 120-150 which was narrowand of greater intensity than the peak observed for the FS-activincomplex in serum. When radiolabelled inhibin was incubated with asimilar concentration of FS as used for activin, a shift ofradioactivity to higher M_(r) was observed, but not in the form of asharp peak as observed for activin. The broad peak elutes at an M_(r) ofapproximately 65-200,000, amounting to only a small amount of theradiolabelled inhibin added, suggesting the affinity of FS is much lowerfor inhibin than for activin.

Selectivity of FS recognition of activin was determined with the solidphase assay described herein. In the assay, the maximal binding ofradiolabelled activin was observed to be 50%, and was obtained with25-50 ng FS adsorbed to the plates. This insures that a majority of FSbinding sites would be occupied by added trace compounds. This increasesthe likelihood that competition, if present, would be detected.Referring to FIG. 4, unlabelled FS inhibits binding of radiolabelledactivin with an ED₅₀ of approximately 1 ng. A similar inhibition curvewith unlabelled inhibin was parallel to that of activin, although thebinding was inhibited with an ED₅₀ of approximately 500 ng, suggesting a500-fold decrease in relative potency. In contrast, the related hormonesTGF-β and MIS did not bind to FS at doses comparable to those used forinhibin. These results demonstrate that FS is selective in itsrecognition of inhibin and activin through the common β_(A) subunit,with activin being greatly preferred, and that both activin and inhibinform complexes with FS.

FS-activin complexes inhibit the biological activity of activin, andreduce activin-stimulated release of FSH. Referring now to FIGS. 5 and7, pituitary cells were cultured using a perifusion column, whichallowed continuous infusion of activin and removal of endogenouslysecreted activin from the culture, and then treated with activin and FS.As seen from the graphs, FS can concentration-dependently block activinstimulation, eventually resulting in the inhibition of FSH biosynthesis.The perifusion system described previously was used to expose cells toactivin or FS alone or combined. When administered alongside of activin,a FS concentration of 10 ng/ml inhibits activin stimulation byapproximately 60% (FIG. 5). Increasing the FS concentration to 30 ng/mlin the same experiment lowers the amount of activin stimulated to lessthan 10% of the level induced in the absence of FS, while higherconcentrations appear to have no additional effect. These resultsindicate the ability of FS to inhibit the biological activity ofactivin, and thus block activin-stimulated release of FSH.

EXAMPLE 2 Synthetic Peptides as Activin Antagonists

FSH biosynthesis can also be inhibited with molecules designed tocompete with activin for the activin receptor, resulting in inhibitionof activin-stimulated release of FSH.

In order to design a molecule capable of competing for the activinreceptor region, the binding between FS and activin (determined by theexperiments described in Example 1) was used to determine a region ofthe activin molecule having biological activity. Substantially pure FSwas characterized in human serum using gel filtration, chromatography,SDS-PAGE/Western blotting techniques, and kinetic analysis. Compoundsfeaturing FS bound to radiolabeled and unlabeled activin were analyzedusing a gel filtration apparatus by observing shifts in molecular weightof labeled activin when bound to FS. Overlapping synthetic peptides wereused to determine two primary domains in the FS-activin binding region,one near the N-terminus and the other near the C-terminus.

Peptides used to map the activin binding region were synthesized usingtechniques described previously (Grausepohl et al., Peptide Chemistry,Structure and Biology, Rivers and Marshall, eds., p. 102, (1990)).Overlapping 15-mer peptides spanning the entire inhibin/activin β_(A)subunit were synthesized on an Abimed 422 automated synthesizer asamides, except for the C-terminal peptide, which was a free acid. Thepurity of each peptide was accessed by HPLC, and confirmation that themajor component of each peptide was the desired sequence was obtained bymass spectrometry.

To be considered active, a peptide had to demonstrate dose-dependentinhibition of activin-FS binding in at least three assays. Referring nowto FIG. 6, three 15-mer synthetic peptides demonstrated consistentinhibition, with the degree of inhibition increasing with increasingconcentration of the peptide. Two of the peptides overlap, indicatingthat the activin binding region contains a minimum of two primarydomains, one contained within the N-terminus (amino acid sequence 15-29of the activin β_(A) subunit: Gln Phe Phe Val Ser Phe Lys Asp Ile GlyTrp Asn Asp Trp Ile (SEQ ID NO:4)) and the other domain near theC-terminus (amino acid sequence 102-113 of the activin β_(A) subunit:Lys Lys Asp Ile Gln Asn Met Ile Val Glu Glu Cys (SEQ ID NO:5)).

Due to similar homology, the recently published crystal structure ofTGF-β was used as a likely model for activin. The structure of thiscompound indicates that the domains are located at opposite ends of thefolded activin molecule. As suggested by the crystal structure, the102-113 sequence of one subunit is juxtaposed to the N-terminus of thedisulfide-linked subunit including the 15-29 sequence.

Synthetic peptides were manufactured as described above and then testedfor the ability to inhibit activin's action at the pituitary by bindingto the activin receptor, thus blocking FSH release. The amino acidsequence of the BIN-3 (SEQ ID NO:3) peptide was composed of the aminoacid sequence 82-114 of the activin β_(A) subunit, derived from thebinding region near the C-terminal end of the activin molecule. Otheramino acid sequences taken from this region, such as 82-98 (BIN-4; SEQID NO:1)), 99-114 (BIN-2; SEQ ID NO:2)), and 66-80 (BIN-1) were alsotested as activin antagonists. Other antagonist candidates can beroutinely screened using this assay.

Initial column chromatography experiments eluting labeled BIN-3 (SEQ IDNO:3) with serum and buffer solutions show evidence for the formation ofan FS-BIN-3 complex, indicating that the BIN-3 (SEQ ID NO:3) amino acidsequence is representative of the activin binding region, and mayfunction as an activin antagonist.

Referring now to FIGS. 8 and 9, BIN-3 (SEQ ID NO:3) inhibits basal andactivin-stimulated FSH secretion when applied to static, plated culturesof rat pituitary cells. Within experimental error, low doses (10-100 ng)of BIN-3 have no effect on the level of FSH. When higher doses (1-10 μg)of BIN-3 are applied to the cell cultures, the FSH level is lowered,with the decrease being about 12% relative to untreated cells, and about27% relative to cells treated with 5 ng activin. Similarly, BIN-2 (SEQID NO:2) lowers the basal FSH level by about 11% in the same assay whenapplied in 10 μg doses (FIG. 9). When added along with 5 ng activin tocells, a 1 μg dose of BIN-3 (SEQ ID NO:3) resulted in about a 12%decline in FSH level when compared to cells treated with activin alone,indicating the ability of BIN-3 (SEQ ID NO:3) to act as an activinantagonist.

Alternatively, non-peptide mimetics having chemical structuressufficiently homologous with the receptor binding region of the humanactivin β subunit can function as activin antagonists. These moleculesare capable of binding to the activin receptor sites and inhibitingactivin-stimulated release of FSH.

EXAMPLE 3 Inhibin as an Activin Antagonist

Inhibin can concentration-dependently block activin simulation of FSHmRNA levels, partly due to competition by inhibin for binding to theactivin receptor.

In order to monitor the inhibitory effects of inhibin, pituitaries fromadult male Sprague-Dawley rats were dispersed and cells were perifusedas described in Example 1. Cells were allowed to adhere to acrylamidebeads loaded into perifusion columns and were perifused at a rate of 325μl/min. with RPMI-1640 containing 2 g/liter NaHCO3, 0.25% BSA, andvarying concentrations of activin and inhibin. Measurements of FSHβ MRNAwere calculated as ratios to GAPDH MRNA.

Using the in vitro perifusion system, equal mass amounts (3 ng/ml) ofactivin and inhibin resulted in a 72% decrease in FSHβ mRNA levelscompared to levels induced by activin stimulation alone. A maximalconcentration of inhibin (30 ng/ml) suppressed FSHβ mRNA levels by 83%.A 10-fold excess of inhibin was necessary to attenuate the effects ofactivin entirely.

Whole cell receptor assays using ¹²⁵ I-labeled activin confirmed thatthe inhibin used in the perifusion experiments competed for activinbinding sites, although with lower affinity. Direct competition at theactivin receptor therefore accounts for part of the activin/inhibinantagonism observed at the level of FSH mRNA.

EXAMPLE 4 Inhibition of Activin signalling by a Modified ActivinReceptor

In another aspect of the invention, the activin receptor can be mutatedso that it is capable of binding activin, but incapable of signalingonce activin is bound. Disrupting the activin signal pathway representsanother mechanism in which activin action can be blocked, andactivin-induced FSH biosynthesis can be inhibited. Receptors mutated assuch might also block the function of normal, wild type activinreceptors if they are capable of dimerization. This dominant negativemechanism has been demonstrated in principle for activin receptors.

Activins have been shown previously to induce mesoderm in embryonicexplants (Symes et al., Development 101: 185-408 (1967)). Using analogywith signal transduction by tyrosine kinase receptors, e.g., thereceptor for fibroblast growth factor (bFGF), experiments which indicatethat a truncated activin receptor will interfere with the function ofendogenous activin receptor by creating an inactive receptor heterodimerwere carried out by Hemmati-Brivanlou et al., Nature, 359: 609-614(1992), the results of which are incorporated herein by reference.

Truncated activin receptors were constructed using a fragment of DNAfrom a wild-type receptor encoding the entire extracytoplasmic domain(including the signal sequence), the transmembrane domain, and 10 aminoacids from the cytoplasmic domain (excluding the serine/threonine kinasedomain). The DNA were entirely free of 5' and 3' untranslated sequences,and were subcloned in pSP64T cells (Kreig et al., Nucleic Acids Res. 12,7057-7070 (1984)). Synthetic RNA encoding the truncated activin receptorwere injected into the animal pole of two-cell embryos from femaleXenopus laevis, and the effects on isolated animal caps werequalitatively assessed by the presence or absence of tissue-specificmolecular markers. These data indicate that the truncated receptorcompletely and specifically blocks the early morphogenetic response ofanimal caps exposed to activin. Early and late molecular markers formesoderm induction are also specifically blocked by the truncatedactivin receptor. In addition, expression of muscle actin, amesoderm-specific gene that is expressed at the end of gastrulation, isalso selectively inhibited in animal caps injected with the truncatedreceptor.

Experimental results also indicate that bFGF induces muscle actin toroughly 10-fold higher levels in animal caps injected with the truncatedactivin receptor compared with control caps. These data show thatactivin antagonizes the mesoderm-inducing capacity of bFGF, and that themutant receptor can effectively inhibit the effects of activin. Thus, byaltering the activin receptor so that binding occurs but signalling isinhibited, the action of activin is selectively blocked.

Methods of Use

The activin antagonists of the invention can be administered to asexually-mature mammal, particularly a human, via one of the traditionalmodes (e.g., orally, parenterally, transdermally, or transmucosally), ina sustained-release formulation using a biodegradable, biocompatablepolymer, or by on-site delivery using micelles, gels and liposomes. Ifinjected, the peptides of the invention are mixed with a balanced saltsolution at pH 7.4-7.5, e.g., 1X phosphate buffered saline at pH 7.4.

Dosages of the antagonists will vary, depending on factors such ashalf-life of the substance, potency, route of administration, and thecondition of the patient. Generally, in order to act as an effectivecontraceptive agent, the antagonist should be administered to thepatient in a dosage of about 10 to 250 μg/kg/day, preferably 50 to 100μg/kg/day.

The foregoing descriptions of preferred embodiments of the inventionhave been presented for purposes of illustration and description. Theyare not intended to be exhaustive or to limit the invention to theprecise form disclosed. The embodiments chosen are described in order tobest explain the principles of the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ValProThrLysLeuArgProMetSerMetLeuTyrTyrAspAspGly                              151015                                                                        Gln                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AsnIleIleLysLysAspIleGlnAsnMetIleValGluGluCysGly                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ValProThrLysLeuArgProMetSerMetLeuTyrTyrAspAspGly                              151015                                                                        GlnAsnIleIleLysLysAspIleGlnAsnMetIleValGluGluCys                              202530                                                                        Gly                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GlnPhePheValSerPheLysAspIleGlyTrpAsnAspTrpIle                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       LysLysAspIleGlnAsnMetIleValGluGluCys                                          1510                                                                          __________________________________________________________________________

What is claimed is:
 1. A method for contraception by inhibiting therelease, biosynthesis, or biosynthesis and release of FSH in a mammal inneed thereof, said method comprising:administering to the mammal acontraceptive agent comprising a peptide up to 70 amino acids in lengthwhich, when complexed with an activin receptor exhibitsactivin-antagonist activity, such that said contraceptive agent inhibitsactivin-stimulated release, biosynthesis, or release and biosynthesis ofFSH in the mammal, thereby resulting in a decrease in fertility andfacilitating contraception in the mammal.
 2. A method for contraceptionby inhibiting release, biosynthesis, or release and biosynthesis of FSHin a mammal in need thereof, said method comprising administering to themammal a contraceptive agent comprising a non-peptide mimetic of thereceptor binding region of the activin β subunit, said non-peptidemimetic displaying activin-antagonist activity by complexing with theactivin receptor and thereby inhibiting activin-stimulated release,biosynthesis, or release and biosynthesis of FSH, resulting in adecrease in fertility in the mammal.
 3. A method for contraception byinhibiting release, biosynthesis, or release and biosynthesis of FSH ina mammal in need thereof, said method comprising administering to themammal a contraceptive agent comprising a peptide havingactivin-antagonist activity, wherein the peptide(1) comprises a sequenceat least 8 amino acids in length which is at least 80% homologous withthe sequence of a portion of the receptor binding region of the activinβ subunit, where any differences between the sequences consist of aminoacid substitutions; and (2) complexes with the activin receptor toinhibit activin-stimulated release, biosynthesis, or release andbiosynthesis of FSH, thereby resulting in a decrease in fertility andfacilitating contraception in the mammal.
 4. The method of claim 3,wherein said portion of said receptor binding region comprises theC-terminal 8 amino acids of said receptor binding region.
 5. The methodof claim 3, wherein said portion of said receptor binding regioncomprises the N-terminal 8 amino acids of said receptor binding region.6. A method of inhibiting release, biosynthesis, or release andbiosynthesis of FSH in a mammal in need thereof, said methodcomprising:administering to the mammal a peptide up to 70 amino acids inlength comprising an amino acid sequence selected from the groupconsisting of: Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr AspAsp Gly Gln (SEQ ID NO:1), Asn Ile Ile Lys Lys Asp Ile Gln Asn Met IleVal Glu Glu Cys Gly (SEQ ID NO:2),and substantially homologous variantsof said sequences.
 7. The method of claim 6, wherein said peptidecomprises the amino acid sequence:Val Pro Thr Lys Leu Arg Pro Met SerMet Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn MetIle Val Glu Glu Cys Gly (SEQ ID NO:3),or a substantially homologousvariant of said sequence.
 8. A method for contraception by inhibitingrelease, biosynthesis, or release and biosynthesis of FSH in a mammal inneed thereof, said method comprising administering to the mammal acontraceptive agent comprising a peptide having activin-antagonistactivity, said peptide being up to 70 amino acids in length andcomprising an amino acid sequence which complexes with activin toinhibit activin-stimulated release, biosynthesis, or release andbiosynthesis of FSH, thereby resulting in a decrease in fertility in themammal.
 9. A method for contraception by inhibiting release,biosynthesis, or biosynthesis and release of FSH in a mammal in needthereof, said method comprising administering to the mammal acontraceptive agent comprising a modified activin receptor havingactivin-antagonist activity, said modified activin receptor forming acomplex with an activin receptor in the mammal, wherein the complexbinds activin and inhibits activin-induced signaling in the mammal,resulting in inhibition of activin-stimulated release, biosynthesis, orrelease and biosynthesis of FSH, and a decrease in fertility in themammal.
 10. The method of claim 9, wherein said complex is a dimer. 11.The method of claim 1, wherein said contraceptive agent is admixed witha pharmaceutically-acceptable carrier substance.
 12. The method of claim1, wherein the peptide comprises a portion of a human activin β subunitand the mammal is a human.
 13. The method of claim 1, wherein thepeptide is at least 8 amino acids in length.
 14. The method of claim 2,wherein the mammal is a human.
 15. The method of claim 2, wherein theactivin β subunit is a human activin β subunit.
 16. The method of claim8, wherein the peptide is at least 8 amino acids in length.
 17. Themethod of claim 8, wherein the peptide comprises a portion of a humanactivin β subunit and the mammal is a human.
 18. The method of claim 9,wherein the mammal is a human.